Isolation, identification and antibiotic resistance profile of thermophilic Campylobacter species from Bovine, Knives and personnel at Jimma Town Abattoir, Ethiopia

Thermophilic Campylobacter species are common cause of animal and human bacterial diseases with growing resistance to antimicrobials. The aim of this study was to determine the prevalence and antimicrobial susceptibility pattern of Campylobacter species from bovine, knives and personnel in Jimma Town, Ethiopia. Faecal samples and carcasses swabs were collected from cattle systematically selected from the annual plan of Jimma Municipal Abattoir. Personnel hand and knife swabs were collected after slaughtering each selected cattle. A cross-sectional study with systematic sampling method was conducted from October 2019 to September 2020 for the isolation, identification and antimicrobial susceptibility pattern of thermophilic Campylobacter species. Isolation and identification of Campylobacter species were performed according to the techniques recommended by the International Organization for Standardization, and in vitro antibiotic susceptibility testing was screened using the standard agar disc diffusion method as recommended by Clinical and Laboratory Standards Institutions. A total of 684 samples (171 samples from faeces, carcasses, knives and personnel hands, were collected independently). The overall prevalence of thermophilic Campylobacterspecies was 5.6% (38/684). Majority of the isolates were from faecal samples (12.9%, n = 22) followed by carcass swabs(4.1% n = 7), knife swabs(3.5% n = 6) and personnel hand swabs(1.8% n = 3). Isolated and identified species of C.jejuni, C. coli and C. lari accounted for 63.2%, 23.7% and 13.2%, respectively. The isolated Campylobacter species were found to be resistant to Cephalothin (100%), Ampicillin (60.5%), Cefotaxime (60.5%), Chloramphenicol (47.4%) and Tetracycline (42.1%). On the other hand, the isolates were susceptible to Nalidixic acid (86.8%), Ciprofloxacin (86.8%), Sulphamethazole (84.2%), Ceftriaxone (78.9%), Clindamycin (68.4%) and Cefixime (65.8%). 84.2% of the isolates showed multi-drug resistance for three-to-six drug classes. All the C. lari isolates were multidrug resistant. All the three isolated species of Campylobacter were resistant to Cephalothin, and most were multidrug resistant. Isolation of Campylobacter species from faecal, carcass, knife and hand swabs revealed possible risk of contamination and exposure to Campylobacter infection of those who consume raw meat. Therefore, enactment of hygienic practices during the slaughtering process, proper handling and cooking of meat and awareness creation on jurisdictional antibiotic usage are required to avoid Campylobacter infection.

Introduction Town Abattoir, source of contamination and level of antibiotic resistance. It also serves as a baseline research to study source of contamination, to consider the level of abattoir hygiene and to initiate regular inspection of abattoirs for Campylobacter infection and antimicrobial resistance patterns.

Study area
The study was conducted at Jimma Municipal Abattoir in Jimma Town. Jimma Town is located in Southwest of Ethiopia, 353 km away from the capital city, Addis Ababa. Jimma Town is surrounded by rural districts that mainly rearing cattle, sheep, goat and poultry. The town is geographically located at 7˚41 0 N latitude, 36˚50 0 E longitude, the average altitude of 1,780 meters above sea level, and commonly characterized as temperate weather with mean annual maximum and minimum temperature of 30˚C and 14˚C, respectively. The annual rainfall ranges between 1138mm and 1690mm [19]. Jimma Town has one municipality abattoir and about 140 meat retailers which receive directly slaughter service from the abattoir. The abattoir gives service for 207,573 people (projected population in 2021) in the town with 50.52km 2 area. Jimma Town Municipal Abattoir gives slaughter services of cattle, sheep and goats. The origin of animal for slaughter is mainly from the surrounding districts of Jimma Zone and other nearby areas.

Study design and period
Faecal samples and carcasses swabs were collected from cattle systematically selected from the annual plan of Jimma Municipal Abattoir in 2019/2020. Personnel hand and knife swabs were collected after slaughtering each selected cattle. Cross sectional study design with systematic sampling method was used. The study was conducted for a year (October 2019 to September 2020) at Jimma Town Municipal Abattoir.

Sample size and sampling
The sample size was calculated using single population proportion formula [20]. Previous bovine thermophilic campylobacter prevalence in Jimma Zone is 12.7% [13], 5% of desired absolute precision and 95% confidence interval. Where, n = required sample size, Pexp = expected prevalence, d = desired absolute precision. The estimated annual plan of cattle slaughtering in 2019/2020 at Jimma Town Municipal Abattoir was 2,500 cattle and every 15 th (K) cattle was selected consecutively as the study subject. Totally, 684 samples were taken from apparently healthy bovine (faecal), their carcasses swab, personnel hand swab and knife swab, 171 samples for each sample type. Swabs from the personnel hands and knives were taken from each selected cattle after slaughtering.

Sample collection and transportation
From each selected bovine, about two gram faecal sample was obtained rectally from untouched glove center using swabber stick and s carcass swabs were taken from surface meat of flank and briskets areas (pooled swabs from both sites) using cotton tipped swabs. Samples from the environment, personnel hands and knives were taken aseptically with sterile cotton tipped swabs. All fecal samples were placed into a sterile screw capped container containing 9 ml Cary-Blair transporting media. In case of swab samples, each cotton wool swab was moistened and rubbed on sites continuously for 30 seconds and transferred to a sterile screwcapped test tube containing 10 ml of Cary-Blair transporting media. Then, the samples were transported to Jimma University College of Agriculture and Veterinary Medicine (JUCAVM) Laboratory using ice box at 2-8˚C. All swabs were processed within 3 hours of collection.

Isolation and identification of thermophilic campylobacter species
Isolation and identification of thermophilic campylobacter species were performed according to the techniques recommended by the International Organization for Standardization [21]. Faecal samples, carcass swabs, knife swabs and personnel hand swabs from transport medium was streaked on to Campylobacter blood free selective agar base called modified Cefoperazone Charcoal Deoxycholate Agar (mCCDA from Oxoid Ltd.) that supplemented with SR155H supplement, and non-blood free Campylobacter selective agar base with campylobacter supplement (Blaserwanger: from HIMEDIA). Streaked plates were incubated at 42˚C in anaerobic jar under a micro-aerophilic atmosphere condition (84% N2, 10% CO 2 , 6% O 2 ) produced from gas generating sachets (Campy-Gen TM; Oxoid Ltd.) for 48 hours and plates with no growth were incubated in a micro-aerophilic condition for additional 24 hours (totally for 72 hrs.).
After two to three days incubation under micro-aerobic condition, mCCDA plates were assessed visually for presence of Campylobacter species colonies. One presumptive Campylobacter colony from each selective agar plate was sub-cultured and tested by standard microbiological and biochemical procedures. Preliminary identification of thermophilic Campylobacter species was performed based on microscopy to see characteristic darting motility by preparing wet smear or saline wet mount, with the iris diaphragm closed effectively to contrast the field. Gram stain was performed to identify the characteristics of Campylobacter, Gram negative with an 'S' shaped and gull wing appearance. Moreover, Oxidase test and Catalase test were performed for the identification of thermo-tolerant Campylobacter genera.
The colonies of thermophilic Campylobacter from blood agar medium were picked up with a sterilized cotton swab and put into small tubes containing storage medium (Brain-heartinfusion broth medium) for identification. Triple sugar iron agar (H 2 S production), Hippurate hydrolysis test, resistance to Cephalothin (30 μg disc) and susceptibility to Nalidixic acid (30 μg) disk were evaluated and interpreted. The parameters formed were based on the basis for the identification of Campylobacter species [21,22].
Three to four morphologically identical colonies of bacteria were picked and suspended in sterile normal saline. Turbidity of the broth culture was measured with turbido-meter with in absorbance reading range of 0.08 to 0.1 at 625nm (equivalent to 0.5 McFarland turbidity standards). A loop full of the bacterial suspension was placed at the center of Muller Hinton agar media (Oxoid, Ltd.) supplemented with 5% sheep blood and evenly spread using sterile cotton tipped applicator. After drying, antimicrobial-impregnated disks were appropriately placed on the surface of the agar using sterile forceps by carefully removing one disk from the cartridge. Eight (8) disks were placed on a 150-mm plate (petri dishes) not closer than 24 mm (center to center) and the rest three on small 60-mm petri dishes on the Muller Hinton agar plate and incubated at 42˚C for 48 hours in anaerobic jar using microaerophilic condition generating sachet (CampyGen™ 2.5L Oxoid Ltd.).
For each thermophilic Campylobacter isolate, the zone of inhibition around each of the 11 antibiotic disks was measured to the closest millimeter. Interpretive criteria [24] for Campylobacter susceptibility testing recommended by the Clinical and Laboratory Standards Institute and zone diameter breakpoints for Enterobacteriaceae were used [25]. Each isolate was classified as susceptible, intermediate, or resistant to each of the 11 antibiotics using the published zone diameter standards for Campylobacter [24][25][26]. Ingredient activity of the disk was checked with standardized reference strain of E. coli (ATCC 25922); sensitive to all the antimicrobial drugs tested by Ethiopian Public Health Institute (EPHI) was used as a control. For each, antimicrobial inhibition zone was measured by using digital Vernier calipers and inhibition zone of each antimicrobial was classified (resistant, intermediate, or susceptible) according to Clinical and Laboratory Standards Institutions [23].

Ethics statement
Ethical approval was obtained from Jimma University College of Agriculture and Veterinary Medicine Minutes of Animal Research Ethics and Review committee. Permission was sought from Jimma Town Municipal Abattoir and informed consent was obtained from individuals providing slaughtering service to take personnel hand and knife swabs.

Data management and analysis
Laboratory data were entered to Microsoft Excel spread sheet, cleaned and transported to STATA_MP version 12. Descriptive statistics was used for calculating percentage and frequency distributions, and odds ratio (OR) was used to evaluate the association of variables at 95%CI and p<0.05 was considered as statistically significant.

Multi-drug resistance pattern of thermophilic campylobacter species isolates
The 11 antimicrobial agents were from nine antimicrobial category, accordingly: Ampicillin Drug resistance was recorded among isolated thermophilic Campylobacter species for at least one antimicrobial agent in two to seven antimicrobial classes. Multi-drug resistance (Campylobacter isolate showing resistance against at least to one antimicrobial agent in three or more antimicrobial classes/category) [27] was observed in all isolated species and C. lari showed higher MDR pattern as compared to C. jejuni and C. coli. The resistance pattern of thermophilic Campylobacter species isolates is summarized in Table 5.

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Resistance profile of thermophilic Campylobacter species from Bovine, Knives and personnel at Abattoir

Discussion
Campylobacteriosis is bacterial, foodborne and zoonotic gastroenteritis disease transmitted to human mainly via undercooked or contaminated meat and animal products. Although poultry is considered as the main source of infection for human, cattle also play substantial role for transmission of the disease in Ethiopia, particularly where consumption of raw beef is a culture [28]. According to Jimma Municipal Abattoir report, majority of the animals slaughtered per day for public sale markets are cattle. In the abattoir, cattle usually inspected before slaughtering and the slaughtered cattle were considered as apparently healthy. However, campylobacteriosis is often asymptomatic and self-limiting, and missed during physical diagnosis. Indeed, C. jejuni and C. fetus are responsible to cause infertility and abortion in cattle [29,30]. In the current study, the prevalence of thermophilic Campylobacter species isolates was (12.9%) in fecal samples followed by (4.1%) in carcasses swabs, (3.5%) in knives swabs and (1.8%) in personnel hand swabs. Increased prevalence of Campylobacter species from fecal sample is because the bacteria colonize mainly small intestine of cattle. Carcasses and other environmental samples acquired the bacterium from contamination mainly during slaughtering process and distribution to butcher shops [31]. Previous study conducted in Jimma Zone urban and rural farm animals also documented comparable prevalence (12.7%) of Campylobacter species from cattle fecal samples [15]. However, higher prevalence of Campylobacter species isolates were reported in studies conducted in Gambella and Gondar, Ethiopia [16,18]. The inconsistency of the prevalence may be ascribed to geographical and environmental variability in the country.  5 Cephalothin (CH-30), 6 Chloramphenicol (C-30), 7 Ciprofloxacin (CIP-10), 8 Sulphamethazole Trimethoprim (SXT-25), 9 Tetracycline (TE-30), 10 Clindamycin (DA-10), 11 Nalidixic Acid (NA-30) https://doi.org/10.1371/journal.pone.0276625.t003

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Resistance profile of thermophilic Campylobacter species from Bovine, Knives and personnel at Abattoir

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Resistance profile of thermophilic Campylobacter species from Bovine, Knives and personnel at Abattoir Table 5. Multidrug-resistance patterns of Campylobacter species isolated from Jimma Town municipal abattoir.

Sub-total (%)
Against two 1  The second highest prevalence of thermophilic Campylobacter species was isolated from carcass/meat swab in this study. Study conducted in Mekele, Northern Ethiopia also reported higher prevalence (11.9%) of Campylobacter species isolates from cattle meat [17]. This could be due to Campylobacter species are the normal flora of gastrointestinal tract [32,33] and in case of carcass swabs, it could be due to contamination of carcass with intestinal contents during manual skinning, evisceration, carcass washing and processing at abattoir. Similarly, Campylobacter species were isolated from hand and knife swabs, and it could be due to cross contamination due to negligence of hand and knife washing in between cattle slaughtering. Personnel hands and utensils such as knife and cutting boards are among significant contributors for the contamination of animal food products with Campylobacter species [3][4][5]. Even though no statistically significant difference was observed, the number of Campylobacter species isolated from knives swabs was higher than personnel hands swabs. Study conducted in Denmark also reported possible Campylobacter species cross contamination of meat products from workers' hands and stabbing knives [34]. Therefore, abattoir management should focus on hygiene of slaughtering houses by avoiding free movement, washing hands and knife after slaughtering each cattle and improving water supply. As well as, implementing regular inspection in abattoirs for infectious diseases including Campylobacter species is mandatory at national level. Moreover, awareness creation or training should be given for abattoir workers to minimize or prevent cross contamination and the risk of zoonotic food borne infectious diseases transmission to human.
The predominant thermophilic Campylobacter species isolate in the current study was C. jejuni followed by C. coli and C. lari, respectively. Previous study conducted in Jimma Zone documented similar findings but the number of C. lari is higher in the current study, 13.2% versus 3.1% [15]. This might be due to increased drug resistance pattern of C. lari observed in this study.
An increasing numbers of Campylobacter isolates have developed resistance to antimicrobials, mainly in low and middle-income countries. In recent years, increased resistance was observed for fluoroquinolones, macrolides, aminoglycosides, and beta lactams [35]. Furthermore, intrinsic resistance in C. jejuni and C. coli has been described against penicillins, most of the cephalosporin as well as trimethoprim, sulfamethoxazole, rifampicin, and vancomycin [36][37][38][39]. Campylobacter isolates in the present study were susceptible to Nalidixic acid (86.8%), ciprofloxacin (86.8%), Sulphamethazole (84.2%) and ceftriaxone (78.9%). However, Campylobacter species in this study were highly resistant for chloramphenicol and Tetracycline. Studies conducted in Morocco also documented resistant strains of Campylobacter species for tetracycline and other regimen of antibiotics [39,40]. Other studies also reported chloramphenicol and Tetracycline resistant Campylobacter species [16,41]. Tetracycline has been used for animal production for many years, and this study also showed high tetracycline resistance in all species of Campylobacter isolates. The resistance to tetracycline in Campylobacter species might be due to presence of plasmid-encoded tet (O) resistant gene in both C. jejuni and C. coli [38,39]. Campylobacter species isolates in this study were also highly resistant to Cephalothin, Ampicillin and cefotaxime. Other studies conducted elsewhere also documented resistance of Campylobacter isolates for these drugs [16,17,42]. This might be alarm for the future serious challenges in the treatment of human campylobacteriosis associated with food of animal origins.
Multidrug resistance was observed in all Campylobacter isolates in the present study. all isolates of C. lari showed particularly, the highest multidrug resistance pattern, against five to six drug classes, followed by C. jejuni, against three to five drug classes. The finding was in agreement with the result documented in Gambella, Ethiopia [16]. Several investigators have also reported the increasing incidence of high multidrug resistance of Campylobacter species [39,43,44]. Multiple drug resistance of thermophilic Campylobacter species might be increased due to transmission of the bacteria from human to animal and vice versa, and these expose the bacterium to different antimicrobial treatments utilized for both animal and human.
The limitation of this study was lack of molecular techniques for identification of Campylobacter species and for the detection of responsible genes for the developed drug resistance. Moreover, we did not used standard strains of Campylobacter species; instead we used E. coli (ATCC 25922) to check the contents of drugs.

Conclusion
Three species of Campylobacter identified were mainly isolated from faecal samples. Most of the isolates were resistant to Cephalothin, Ampicillin and Cefotaxime. Multidrug resistance among the isolates was also high. Detection of Campylobacter species from the faeces, carcass, knife and personnel shows possible cross-contamination during the slaughtering process. This may increase the risks of Campylobacter infection to people consuming uncooked meat. High proportion of multidrug resistance among the isolates may also result in horizontal spread of drug resistance in human and may pose a threat to humans and further limits therapeutic options. Based on the findings; it is sensible to recommend regular coordinated actions like judicial antibiotic usage and hazard analysis of critical control points from the farm, through the abattoir to the retailer and awareness creation on good hygienic practice and processing of raw meat to minimize or eliminate the risk of infectious agents in general and Campylobacter contamination in particular.